Combined cerium and zinc oxide nanoparticles induced hepato-renal damage in rats through oxidative stress mediated inflammation

The toxicity profiles of nanoparticles (NPs) used in appliances nowadays remains unknown. In this study, we investigated the toxicological consequences of exposure to cerium oxide (CeO2) and zinc oxide (ZnO) nanoparticles given singly or in combination on the integrity of liver and kidney of male Wistar rats. Twenty (20) rats were allotted into four groups and treated as: Control (normal saline), CeO2NPs (50 μg/kg), ZnONPs (80 μg/kg) and [CeO2NPs (50 μg/kg) + ZnONPs (80 μg/kg)]. The nanoparticles were given to the animals through the intraperitoneal route, three times per week for four repeated weeks. Results revealed that CeO2 and ZnO NPs (singly) increased serum AST and ALT by 29% & 57%; 41% & 18%, and co-administration by 53% and 23%, respectively. CeO2 and ZnO NPs increased hepatic and renal malondialdehyde (MDA) by 33% and 30%; 38% and 67%, respectively, while co-administration increased hepatic and renal MDA by 43% and 40%, respectively. The combined NPs increased hepatic NO by 28%. Also, CeO2 and ZnO NPs, and combined increased BAX, interleukin-1β and TNF-α by 45, 38, 52%; 47, 23, 82% and 41, 83, 70%, respectively. Histology revealed hepatic necrosis and renal haemorrhagic parenchymal in NPs-treated rats. Summarily, CeO2 and ZnO NPs produced oxidative injury and induced inflammatory process in the liver and kidney of experimental animals.

one of the essential nanoparticles having applications in fuel cells, oxygen pumps, gas sensors, and as a fertilizer in Chinese agriculture 15 . The self-generating ability of cerium oxide nanoparticles by reversibly inter-switching between oxidation states of + 3 and + 4 renders it an important therapeutic agent. In contrast to other nanoparticles, the cerium oxide in nano state maintains its fluorite configuration, thereby forming an oxygen vacancy in its lattice 16 . The vacant oxygen so form renders it more catalytically active and thus its antioxidant potentials 17 . The biomedical application of cerium oxide nanoparticles includes its ability to reduce tumor volume and weight in experimental rats as well as its cytotoxic, pro-apoptotic, and anti-invasive capacity on melanoma cells 18 . The mechanism of antitumor activity of cerium oxide nanoparticles has been traced to its antioxidant activity against reactive oxygen species (ROS) 19 . Recent studies have found strong association between exposure to ultra fine nanoparticles and adverse health effects in humans 20,21 . Due to their unique physical and chemical characteristics, nanoparticles have been the focus of much research owning its industrial applications and environmental toxicity 22 . The biomedical and industrial applications of these nanoparticles has therefore increased the possibility of human exposure to more than one nanoparticles per time and their resultant effect might posse more harm than good to public health. Therefore, this study was designed to evaluate the toxic effects of cerium and zinc oxide NPs, given singly or combined on the hepato-renal system of male Wistar rats.

Materials and methods
Chemicals. Glutathione  Nanoparticles. The nanoparticles (CeO 2 NPs) and (ZnO NPs) used during this study were obtained from Greg Goss Research Group, University of Alberta, Edmonton, Canada. The cerium oxide and zinc oxide nanoparticles are negatively charged with an average diameter less than 10 nm and 13 nm respectively. They were functionalized with poly acrylic acid polymer coating to render it highly dispersible even in suspension. The unique features of the particles in solution have been highly characterized and described by 23 . Ethical approval. Before the commencement of this study, approval was obtained from the Animal Care and Use Regulatory Committee (ACUREC), University of Ibadan, Nigeria and the body endorsed the experimental procedures with approval number UI-ACUREC/18/0039. The study was carried out in accordance with relevant guidelines and regulations in handling of experimental animals, where they were treated with the least discomfort. The study is reported in accordance with ARRIVE guidelines.

Treatment of animals.
Mature male Wistar rats (140.00 ± 5.20 g) were acquired from Animal breeding division of the Faculty of Basic Medical Sciences, University of Ibadan, Nigeria. The animals were housed in properly aerated cages and maintained at ambient temperature (25 °C) under a regulated 12:12 light/dark phases. They were nourished with standard laboratory feed and water ad libitum. Experimental design. The animals were allowed to acclimatize for two weeks and thereafter, twenty (20) rats were assigned into four groups of five animals each. The first group (control) received normal saline, while other groups received cerium oxide nanoparticles (CeO 2 NPs) at 50 μg/kg body weight, zinc oxide nanoparticles (ZnONPs) at 80 μg/kg body weight and [CeO 2 NPs (50 μg/kg) + ZnONPs (80 μg/kg) ] via intraperitoneal route. The route and dosage were decided according to the studies of 24,25 , respectively. The nanoparticles were given thrice per week for four consecutive weeks. The reason for administering thrice per week and not daily was to prevent bioaccumulation of the nanoparticles which may contribute to toxic responses that will be beyond the scope of the research. The choice of 50 μg/kg CeO 2 NPs was due to the previous result from our laboratory where we used graded doses (100 μg/kg, 200 μg/kg and 300 μg/kg) of CeO 2 NPs and result showed that these doses induced organ toxicity Adebayo et al. (2018). We therefore decided to check if exposure to lower doses would be safe. The reason for choosing 80 μg/kg ZnO NPs dose?
Preparation of tissue and serum. Twenty-four hours after the administration of last dose of CeO 2 NPs and ZnONPs, food was withdrawn from the animals overnight, anesthetized (using ketamine at 35 mg/kg) and sacrificed by cervical dislocation the next day. The liver and kidney were carefully excised, rinsed in ice cold buffer solution (1.15% KCl) and blotted. The weight of the organs was determined and recorded. Part of the whole liver was cut, and one of the pair of the kidneys were kept in 10% buffered formalin. The remaining portions of liver and whole kidney were homogenized in phosphate buffer (50 mM & pH 6.8). The resulting mixture was centrifuged at 10,000g for 15 min to obtain post-mitochondrial fraction (PMF). Blood was collected from rats via retro-orbital bleeding into plain bottles, allowed to clot and centrifuged at 3000g for 15 min to obtain serum. Both serum and post mitochondria fraction, PMF were used for biochemical analyses.

Biochemical assays. Determination of alanine and aspartate aminotransferases (ALT & AST), and total
bilirubin. Activities of ALT, AST and level of total bilirubin in the serum were evaluated using the method described by [26][27][28]  www.nature.com/scientificreports/ mixture. This was followed by thirty minutes of incubation at a temperature of 37 °C. Later on, a volume of 500 µL of 2, 4-dinitrophenylhydrzine was introduced into the reacting mixture and left on the bench for about 20 min at a temperature of 25 °C. An additional (5.0 mL) NaOH was introduced, and then the optical density (546 nm) was recorded alongside the (reagent) blank within 5 min.
Alanine aminotransferase activity. Diluted sample (100 µL) was reacted with l-alanine (0.1 mL), sodium phosphate buffer solution (0.1 mL of 100 mmol/L at 7.4 pH), and 2 mL of α-oxoglutarate to make the reaction mixture. This was followed by 30 min of incubation at a temperature of 37 °C. Later on, a volume of 500 µL of 2, 4-dinitrophenylhydrzine was introduced into the reaction mixture and left on the bench for about 20 min at a temperature of 25 °C. An additional (5.0 mL) NaOH was introduced, and then the optical density (546 nm) was recorded alongside the (reagent) blank within 5 min.
Determination of total bilirubin. Briefly, the reaction mixture contained 200 μL of sample, 200 μL of a solution of sulfanilic acid (29 mmol/L) and hydrochloric acid (0.17 N), 50 μL of sodium nitrite (38.5 mmol/L), and 1.0 mL of caffeine (0.26 nmol/L) and sodium benzoate (0.52 mol/L), which were incubated at room temperature for 10 min. After incubation, 1.0 mL of tartrate (0.93 mol/L) and sodium hydroxide (1.9 N) were added and the mixture was incubated for 10 min at room temperature. The absorbance was read against a blank at 546 nm.
Determination of urea and creatinine levels. Serum urea and creatinine levels were assessed by the methods of 29,30 respectively.
Protein determination. Serum and PMF protein levels were determined according to the method of 31 using bovine serum albumin (BSA) as standard. In brief, 0.5 mL diluted sample/ standard was added to 0.7 mL Lowry reagent [Mixture of alkaline solution (NaOH and Na 2 CO 3 )] and was incubated for 20 min at room temperature. Thereafter, 0.1 mL of diluted Folin's reagent (2N Folin and Ciocalteu's phenol reagent, and 6 mL distilled water) was added, vortex mixed and incubated for 30 min. After incubation, absorbance was taken at 750 nm and protein values were estimated by extrapolation from BSA calibration curve.
Determination of catalase (CAT) activity. Catalase activity was determined by the method of 32 . The mixture of 2.4 mL of phosphate buffer (50 mM, pH 7.0), 1.0 mL of 19 mM H 2 O 2 and 50 µL of sample were incubated at room temperature for 3 min. Next, the reaction was terminated by the addition of 2 mL of dichromate/acetic acid solution, followed by heating in boiling water bath for 10 min. The solution was cooled at room temperature and, the decrease in absorbance was measured in a spectrophotometer at 570 nm for 3 min. Catalase activity was expressed as Unit/mg protein.
Assessment of the activity of superoxide dismutase (SOD). Activity of SOD was determined according to the method of 33  Estimation of the activity of glutathione-s-transferase (GST). The activity of GST was determined by the method of 34 using CDNB as a substrate. The reaction mixture is made up of 1.7 mL of 100 mmol/L phosphate buffer (pH 6.5) and 0.1 mL of 30 mmol/L of CDNB. After pre-incubating the reaction mixture at 37 °C for 5 min, the reaction was started by the addition of 20 µL of sample, and absorbance was followed for 5 min at 340 nm. Reaction mixture without the enzyme served as a blank. Specific activity of GST was expressed as micromole of GSH/ CDNB conjugate formed per min per mg protein using an extinction coefficient of 9.61 per mmol per cm.
Assessment of the activity of glutathione peroxidase (GPx). The GPx activity was determined by the method of 35 The reaction mixture contained 500 µL of sodium phosphate buffer, 100 µL of 10.0 mM of sodium azide, 200 µL of 4.0 mM of reduced glutathione (GSH), 100 µL of 2.5 mM of H 2 O 2 and 50 µL of the sample. This was made up to 2.0 mL with distilled water and incubated for 3 min at 37 °C. The reaction was terminated by the addition of 0.5 mL of 10% trichloroacetic acid and centrifuged. The supernatant obtained was used for the determination of residual GSH by the addition of 4.0 mL of disodium hydrogen phosphate (0.3 mM) solution, and 1.0 mL of 5,5''-dithio-bis-2-nitrobenzoic acid (DTNB) reagent. The absorbance was measured in a spectrophotometer at 412 nm and GPx activity was expressed as micromole/mg protein.
Estimation of reduced glutathione (GSH) level. Level of GSH was determined according to the method of 36 .
Briefly, an aliquot of PMF was deproteinized by the addition of an equal volume of 4% sulfosalicylic acid, and the resulting solution was centrifuged at 3000g for 15 min at 4 °C. Supernatant (100 µL) was then added to 1.5 mL of DTNB. The GSH was proportional to absorbance at 412 nm. Values were expressed in micromole/g tissue.
Assessment of lipid peroxidation level. The amount of lipid peroxide formed in the liver and kidney was estimated by the method of 37  www.nature.com/scientificreports/ at 3000g. The optical density of the supernatant was measured using a spectrophotometer at 532 nm against a blank.
(b) Estimation of nitric oxide (NO) level. The concentrations of NO 3 − and NO 2 − in the serum, liver and kidney were estimated as an index of NO production by the method of 38 . The amount of nitrite in the serum, renal and hepatic PMF was estimated following Griess' reaction. In the reaction mixture, 0.5 mL of sample was incubated with 0.5 mL of Griess' reagent at 37 °C for 20 min. The absorbance was measured in a spectrophotometer at wavelength 550 nm, and the concentration of nitrite was assessed by comparing the resulting optical density with a standard curve of well-known sodium nitrite concentration.
(c) Estimation of the activity of myeloperoxidase (MPO). The activity of myeloperoxidase (MPO) was assessed by the modified method of 39 . The hepatic and renal MPO activities were estimated using spectrophotometry technique by reacting O-dianisidine with hydrogen peroxide. Myeloperoxidase, a lysosomal enzyme catalyzes the oxidation of O-dianisidine in the presence of H 2 O 2, acting as an oxidizing agent to yield a brown-coloured product, which absorbs maximally at wavelength 470 nm.

Statistical analysis.
The results were presented as mean ± standard deviation. Data were analyzed by oneway analysis of variance (ANOVA) using SPSS version 23.0 (SPSS Inc., Chicago, IL, USA). For the post Hoc test, the Least Significant Difference (LSD) was used and the differences between control and test groups were considered significant at p-values less than 0.05. Table 1 showed that rats administered CeO 2 and ZnO NPs singly or combined had insignificant (p > 0.05) increase in the organo-somatic weight of liver relative to the control group. The activities of serum AST and ALT significantly (p < 0.05) increased in animals administered CeO 2 and ZnO NPs singly as well as the combined group (Table 2). Precisely, AST activities increased by 29%, 59% and 53% in rats administered CeO 2 and ZnO NPs, and combined group, respectively. Similarly, ALT activities increased by 41% and 23% in rats administered CeO 2 NPs and [CeO 2 NPs + ZnONPs], respectively.    www.nature.com/scientificreports/ rats administered CeO 2 and ZnO NPs singly, and in combined group. Likewise, the levels of serum lipid peroxidation products increased by 34%, 36% and 48% in animals given CeO 2 and ZnO NPs singly, and combined group, respectively. In addition, levels of lipid peroxidation in the serum increased by 33%, 34% and 47% in animals given CeO 2 and ZnO NPs singly, and combined group respectively. (Fig. 2). However, insignificant (p > 0.05) decrease in the levels of serum total sulphydryl was observed in the NPs administered groups when compared to the control, with the exception of ZnO NPs where there was 33% decrease in levels of total sulphydryl. Furthermore, hepatic NO increased by 40, 36 and 38% in rats administered CeO 2 and ZnO NPs singly, and combined group, respectively, while renal NO increased by 37% in animals given CeO 2 NPs alone (Fig. 3). In Fig. 4, activities of renal MPO increased by 98 and 46% in rats administered CeO 2 and ZnO NPs singly, respectively, while hepatic MPO increased by 76% in animals given CeO 2 NPs alone. In Figs. 5 and 6, administration of CeO 2 and ZnO NPs singly, and combined for four consecutive weeks caused significant (p < 0.05) decrease in the activities of renal and hepatic SOD and catalase of the animals. The decrease in the activities of these first line antioxidant enzymes was accompanied by significant (p < 0.05) increase in the levels of renal lipid peroxidation (LPO) products by 40, 118 and 46% and hepatic LPO by 48, 52 and 58%, respectively in animals given CeO 2 and ZnO NPs singly, and combined (Fig. 7). In addition, Figs. 8 and 9 showed that antioxidant parameters; hepatic and renal activities of GPx and the levels of GSH were significantly (p < 0.05) decreased in animals administered  www.nature.com/scientificreports/ these NPs either singly or in combination. In addition, inflammatory markers; IL-1β and TNF-α increased by 47, 23, 82% and 41, 83, 70% in rats treated with CeO 2 and ZnO NPs singly, and combined, respectively ( Table 3). Table 3, administration of CeO 2 and ZnO NPs singly and combined, increased the levels of serum BAX (Marker of apoptosis) by 45, 38 and 52%, respectively. Figure 10a, b showed insignificant (p < 0.05) increase in the levels of caspases-3 and-9 in rats administered CeO 2 and ZnO NPs singly and combined. Figures 11 and 12 show representative photomicrographs of liver and kidney of rats administered CeO 2 and ZnO NPs singly and combined. In the controls, the tissues displayed normal architectural arrangement with little or no observable lesions. However, in rats treated with CeO 2 and ZnO NPs singly, and combined, hepatic necrosis and infiltration of inflammatory cells were observed. These observations were more pronounced in animals treated with ZnONPs and combined group. Also, rats given CeO 2 and ZnO NPs singly, and combined showed tubular degeneration in renal medulla and congestion of interstitial blood vessel.

Discussion
This study confirmed via biochemical and histological means that CeO 2 and ZnO nanoparticles induced inflammatory and oxidative responses in the liver and kidney of rats. After ingestion and uptake, they are bio-distributed and accumulated in major organs such as the liver, spleen, and kidney 41 , posing systemic toxicity to animals. The  In this present study, the integrity of the liver and kidney was compromised following administration of CeO 2 and ZnO NPs. The damaging effect triggered by the administration of these nanoparticles was established via histopathological examination where alteration in cyto-architecture and infiltration of inflammatory cells in the liver and kidney of animals were observed. There are several mechanisms of hepatic and renal toxicity induced by nanoparticles, these include generation of ROS/free radicals, inflammatory processes and apoptosis 44 . In particular, metal-based nanoparticles have been found to induce oxidative stress due to generation of ROS in cells 44 . ROS which are oxygen ions or oxygen-containing radicals have been implicated in cytotoxicity and genotoxicity induced by toxicants, and nanoparticles have been known to play similar roles 45 . The significantly high levels of serum, hepatic and renal malondialdehyde (MDA) and very low levels of total sulphydryl (TSH) observed in NPs-treated rats confirmed induction of oxidative stress in the animals. These results are in tandem with the findings of 46 who reported alteration in homeostasis of total sulphydryl and lipid peroxidation in kidney of experimental rats, and 44 who showed that certain nanoparticles exert toxicity through the production of ROS such as superoxide anion, hydroxyl radicals and hydrogen peroxide. Superoxide dismutase (SOD) and catalase are first-line antioxidant enzymes against ROS. SOD is involved in the scavenging of superoxide anion radicals by converting them to hydrogen peroxide and oxygen, thus preventing peroxynitrite production and further damage 47 . In this study, the observed oxidative stress in the liver and kidney of nanoparticle-treated rats  www.nature.com/scientificreports/ was accompanied by a significant decrease in hepatic and renal activities of SOD, catalase and glutathione peroxidise, and depletion of reduced glutathione. Decrease in antioxidant status of animals is a predisposing factor to the development of diseases and infections 48 . The enzyme: nitric oxide synthase (NOS) catalyzes the reaction that produces nitric oxide (NO) from l-arginine and oxygen 49 . As a vasodilator, nitric oxide is thought to play an important role in the homeostatic modulation of renal hemodynamics in both normotensive and hypertensive states 50 . Imbalance in redox homeostasis via elevated levels of ROS and reduced activities of antioxidant enzymes have been implicated in apoptosis through oxidative damage to intracellular proteins and DNA 51 . In addition, ROS scavenge nitric oxide, leading to the formation of peroxynitrite, which reduces NO bioavailability and induces oxidative damage in biomolecules 52 , all of which contributes to exacerbating inflammation in the tissues. In this study, inflammation of the liver and kidney was confirmed in rats treated with CeO 2 and ZnO nanoparticles singly or combined, suggesting that the NPs can activate key processes, leading to inflammation. Various pro-inflammatory proteins also cause neutrophils to generate myeloperoxidase (MPO) and ROS during bacterial infection 53 . At the site of inflammation, activated neutrophils, monocytes, and tissue macrophages release MPO, which utilizes hydrogen peroxide to oxidize a variety of substrates, including pseudohalides to generate powerful oxidant like HOCl 54 . Elevated MPO, IL-1β and TNF-α observed in the nanoparticles-treated rats in this study further attest to induction of inflammation in tissues of the animals. Pharyngeal aspiration of ZnO-NPs induced infiltration of inflammatory cells in the lung of mice, but minimally induced Nrf2-dependent antioxidant enzymes 40 and acute pulmonary exposure to CeO 2 NPs causes oxidative stress, inflammation, and DNA damage in multiple major organs, including the lung, heart, liver, kidney, spleen, and brain 59 . Caspase-9 is involved in the regulation of physiological cell death and degradation of worn out and damage cells. It is an important protein in the intrinsic pathway of apoptosis where it acts an initiator 55 . Serum activities  www.nature.com/scientificreports/ of caspases-3 and -9 were not significantly altered in rats administered CeO 2 and ZnO nanoparticles either singly or when combined. However, the increase in BAX in rats treated with CeO 2 and ZnO nanoparticles singly and combined might suggest the induction of apoptosis. The Bcl-2 proto-oncogene family is known to regulate apoptosis 56 . Bcl-2 family members exist in two categories: the inhibitor of apoptosis (Bcl-2) and inducer of apoptosis (BAX) 56 . It is the relative abundance of pro-apoptotic and anti-apoptotic proteins that determines the Figure 9. Effect of cerium and zinc oxide nanoparticles on hepatic and renal levels of reduced glutathione in male Wistar rats showing significant decrease in hepatic reduced glutathione in groups administered with cerium oxide nanoparticles singly and combined with zinc oxide, while renal reduced glutathione decreased only in the zinc oxide nanoparticles group. *Significantly different from control (p < 0.05). CeO2NPs cerium oxide nanoparticles, ZnONPs zinc oxide nanoparticles. www.nature.com/scientificreports/ susceptibility to cell death 57 . Our inability to determine the Bcl-2 levels in this study is one of the limitations and it is due to limited fund, as this research is self-sponsored.. In line with our results 58 , reported that CeO 2 nanoparticles prevents the loss of mitochondria membrane potential, thereby preventing the apoptotic loss of cell viability. Due to their physico-chemical properties, nanoparticles have been found to invade tissues and organs (liver and kidney inclusive) through dermal exposure 40 . After nanoparticles have been administered, they can bio-accumulate in the liver, and followed by the kidney 40 . This necessitated to examine the effects of the two nanoparticles on the cytoarchitecture of the liver and kidney of the animals. In the liver of animals exposed to CeO 2 nanoparticles, the sinusoids show mild infiltration of inflammatory cells and focal area of hepatocytes with necrosis (10-30%), while the moderate portal congestion and mild periportal infiltration of inflammatory cells observed in animals exposed to ZnO nanoparticles (Table 4) and the combination of CeO 2 and ZnO is indicative of the ability of both nanoparticles. This observation was in tandem with Sabry et al. who reported that microscopic examination of histological sections of the liver showed pathological changes after injection of mice with cerium oxide in both low and high doses included dilatation in the central vein, congestion, infiltration of inflammatory, hyperplasia of hepatic cells, multinucleated cells and necrosis 60 . The control group showed intact hepatic architecture. The kidney parenchyma of animals exposed to CeO 2 nanoparticles showed focal area of mild haemorrhage (6-10%) and fluid accumulation and the interstitial spaces show interstitial vascular congestion (Table 5) and moderate infiltration of inflammatory cells (scattered). In the ZnO nanoparticle treated rats, the interstitial spaces show moderate congestion (scattered), while in the combined nanoparticles group, the renal cortex showed glomeruli with dilated capsular spaces. The present study confirmed earlier studies on the toxicity and adverse effect of exposure to CeO 2 and ZnO nanoparticles, and also showed for the first time that exposure to the combined nanoparticles triggered hepatorenal toxicities via imbalance in oxidant /antioxidant levels. These effects caused oxidative stress and inflammation in the rats leading to alteration in the cyto-architecture of the liver and kidney.   Figure 11. Photomicrograph showing the effect of cerium and zinc oxide nanoparticles on the integrity of hepatocytes in male Wistar rats. In the control, the sinusoids appear normal (slender arrow) without infiltration of inflammatory cells, CeO2 NPs and ZnO NPs groups shows necrosis (reds arrow)] and infiltration of inflammatory cells (blue arrow) respectively, while in the ZnO NPs + CeO2 NPs group, the liver parenchyma show severely dilated sinusoid with congestion (green arrow).  Figure 12. Photomicrograph showing the effect of cerium and zinc oxide nanoparticles on the integrity of renal tissues in male Wistar rats. In the control, the renal cortex shows normal glomeruli with normal messengial cells and capsular spaces (black arrow), while in CeO2 NPs group, the interstitial spaces shows mild haemorrhage (red arrow). In the ZnO NPs group, the renal cortex show severe periglomerular infiltration of inflammatory cells (red arrow) while ZnO NPs + CeO2 NPs group, the interstitial spaces show moderate congestion (green arrow).  www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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